1. Field of the Invention
This invention relates to novel growth hormone, especially bovine growth hormone, muteins which inhibit the growth of animals or otherwise antagonize the effects of endogenous growth hormone. These analogues may be expressed in transgenic animals which thereby acquire a "reduced growth" phenotype.
2. Information Disclosure Statement
Bovine growth hormone (GH) is a protein of 191 amino acids that is naturally synthesized in the anterior pituitary. The molecular weight of the mature protein is about 22,000 daltons, but it is initially made as a pre-growth hormone with an extra 26 amino acids on the amino terminal. This leader (or signal peptide) is normally cleaved during secretion of the hormone by bovine pituitary cells. Several forms of the mature protein have been found in nature. The N-terminal can vary (due to variation in the site of cleavage during secretion) so that the mature protein begins with either NH.sub.2 -Ala-Phe-Pro or NH.sub.2 -Phe-Pro. Additionally, the amino acid at position 126 may be either leucine or valine, apparently as a result of allelic variation in the bovine population.
Exogenous administration of bGH to cattle increases milk production, feed efficiency, growth rate, and the lean-to-fat ratio, and decreases fattening time.
bGH has been produced by recombinant DNA techniques, see e.g., Fraser, U.S. Pat. No. 4,443,539 (yeast; Buell, EP Appl. 103,395 (bacteria); Krivl, EP Appl. 193,515 (bacterial); Kopchick, EP Appl. 161,640 (encapsulated mouse cells implanted into animals); DeBoer, EP Appl. 75,444 (bacteria; gene modified to eliminate harmful secondary structure) and this has facilitated the production of analogues of bGH by site-specific mutagenesis. Thus, Aviv, GB 3,073,245 describes production of Met Pro (des Ala) bGH, Met Arg (des Ala) bGH, Met-Glu-Gly (des Ala) bGH, and des (Ala.sup.1 Phe.sup.2 -Pro.sup.3 -Ala.sup.4) bGH in E. coli. Brems, et al., PNAS (USA) 85:3367-71 (1988) reported preparation of the bGH mutant K112L, which extended the hydrophobic face of the third alpha helix of bGH. The 96-133 fragment of this mutant was also prepared.
The biological activity of proteolytic fragments of bGH has also been studied. Brems, et al., Biochemistry, 26:7774 (1987); Swislocki, et al., Endocrinology, 87:900 (1970); Paladini, et al., TIBS, 256 (Nov. 1979). The fragment of bGH containing amino acids 96-133 is superior in growth promoting assays to bGH 1-95 and bGH 151-191. Hara, et al., Biochemistry, 17:550 (1978); Sonenberg, U.S. Pat. Nos. 3,664,925 and 4,056,520; Chen and Sonenberg, J. Biol. Chem., 250:2510-14 (1977). An octapeptide derived from the amino-terminal of hGH has been shown to have hypoglycemic activity, see Ng, et al., Diabetes, 23:943-949 (1974), but it has no effect on growth. Similar results were observed with the fragment bGH (96-133). Graf, et al., Eur. J. Biochem., 64:333-340 (1976); Hara, et al., Biochem., 17:550-56 (1978).
Analogues of bGH have varied in growth-promoting activity, as have the known analogues of other growth hormones. However, a growth hormone analogue having growth-inhibitory activity has not previously been reported.
A variety of transgenic animals have been produced. Hammer, et al., Nature, 315:680-683 (1985) (rabbits, sheep and pigs). Certain of these animals have been caused to express a growth hormone, and increased growth of such transgenic animals has been reported. Palmiter, et al., Nature 300:611 (1982) microinjected the male pronucleus of fertilized mouse eggs with a DNA fragment containing the promoter of the mouse metallothionein-I gene fused to the structural gene of rat growth hormone. Several of the transgenic mice developed from the genetically modified zygote exhibited a growth rate substantially higher than that of control mice. (In effect, the genetically modified mouse serves as a test environment for determining the effect of the hormone on animal growth). Later, Palmiter, et al., Science, 222:809 (1983) demonstrated that a similar enhancement of growth could be obtained in transgenic mice bearing an expressible human growth hormone gene. A like effect is observed when human growth hormone releasing factor is expressed in transgenic mice. Hammer, et al., Nature, 315:413 (1985).
Bovine growth hormone has also been expressed in transgenic animals. McGrane, et al. J. Biol. Chem., 263:1144351 (1988); Kopchick, et al., Brazil. J. Genetics, 12:37-54 (1989). However, transgenic animals characterized by an exogenous gene which confers a reduced growth phenotype were hitherto unknown.